Contents

A. Primer design for PCR

For typical BioBricks construction, you want a PCR product which has an XbaI site upstream of your part, and SpeI, NotI, and PstI sites downstream of your part. Then, the forward primer should be of the form: 5' CCTTTCTAGA (15-20 bp complement to coding strand) 3' and the reverse primer should be of the form: 5' AAGGCTGCAGCGGCCGCTACTAGT (15-20 bp reverse complement) 3'

Design considerations. Make sure that:

The Tm of each primer is between 55-65 °C

The Tm of both primers are very similar, i.e., within ~2 °C

Either primer will not form a stable internal hairpin structure, i.e., ΔG <-3 kcal/mol

The forward and reverse primers do not combine to form a stable hairpin structure or dimer

The genomic DNA to be amplified does not contain any EcoRI, PstI, SpeI, XbaI, or AarI sites

The insert for the forward primer does not begin with TC (or else a DAM I site (GATC) is formed, and XbaI cannot cut)

Under the Primer tab, set "Before" and "After" to 0 bp. Adjust the Tm, primer length, GC content, et c. as noted above. Also click More>> and insert the flanking sense and anti-sense sequences (given at top) in the boxes "Attach to 5' terminus of Sense primer" and "Attach to 5' terminus of Anti-sense primer". Lastly, click "Apply" then "OK."

Three possible sets of designed primers will appear in a folder on the left side of the screen, ranked by their score. Usually, the best possible score is 171. The lowest score that I (Caroline) have successfully used is ~120.

Double-check your primers by highlighting a given sequence, then right-clicking and selecting "Analyze".

For more information, check out Vector NTI's user manual, Chapters 8 and 20.

Note: if it is not possible to make a good set of primers with the flanking regions described above, see if using a different set of flanking regions improves the primers. For example, you can also use a PCR product that has the EcoRI, NotI, and XbaI sites upstream of your part, while the SpeI site is downstream of your part. In this case, the forward primer would be of the form: 5' CCTTGAATTCGCGGCCGCATCTAGA (15-20 bp complement to coding strand)3' and the reverse primer should be of the form: 5' AAGGACTAGT (15-20 bp complement to coding strand) 3'.

B. PCR from yeast chromosomal or plasmid template

Both are known to work.
My two cents (Caroline): Using Vent (condition A) works for most (>90%) parts. However, there have been few parts for which I couldn't get pcr products using condition A. I have been able to pcr out these difficult parts using Pfx (condition B) -- Pfx has worked for *all* pcr reactions I've tried.

Condition A: Vent polymerase

Resuspend each primer in Tris buffer pH 8.0 or distilled water to 100 µM.

Mix:

5 µL 10x ThermoPol buffer

0.4 µL 25 mM dNTPs

0.5 µL 100 µM forward primer

0.5 µL 100 µM reverse primer

≤1 µL plasmid DNA or 2 µL genomic DNA

1 µL Vent DNA polymerase

distilled water to 50 µL total volume

PCR program:

Start: 95 °C for 2 min. (melt)

Cycle 95 °C for 0.5 min (melt)

Tm minus 5 °C for 0.5 min. (anneal)

74 °C for (# bp/1000) min. (extension) - no less than 0.5 min.

No. of Cycles: 30

End: keep at 4 °C forever

Condition B: Pfx Polymerase

Resuspend each primer in Tris buffer pH 8.0 or distilled water to 100 µM.

Use Stratagene's Pfx kit.

Mix:

3 µL primer mix (10mM of each primer)

0.8 µL template DNA

25 µL 10X PFx amplification buffer

3 µL 10mM dNTPs

2 µL 50mM MgSO4

30 µL 10X PFx enhancer buffer

34.2 µL water (to 100 µL)

2 µL PFx DNA polymerase

PCR Program

Start: 94 °C for 5 min. (melt)

Cycle 94 °C for 15 sec (melt) (cycle start)

55 °C for 0.5 min. (anneal)

68 °C for 3.5 min (extension) (cycle end)

68 °C for 7 min

No. of Cycles: 35

End: keep at 4 °C forever

C. Gel analysis and purification of PCR products

Run 2 µL of PCR reaction on an appropriate percentage agarose gel.

If only your desired band is present, purify using Qiagen's PCR purification kit and elute with 30 µL.